Antibacterial and Biocompatible Polyethylene Composites with Hybrid Clay Nanofillers

Low-density polyethylene is one of the basic polymers used in medicine for a variety of purposes; so, the relevant improvements in functional properties are discussed here, making it safer to use as devices or implants during surgery or injury. The objective of the laboratory-prepared material was to study the antimicrobial and biocompatible properties of low-density polyethylene composites with 3 wt. % hybrid nanoclay filler. We found that the antimicrobial activity was mainly related to the filler, i.e., the hybrid type, where inorganic clay minerals, vermiculite or montmorillonite, were intercalated with organic chlorhexidine diacetate and subsequently decorated with Ca-deficient hydroxyapatite. After fusion of the hybrid nanofiller with polyethylene, intense exfoliation of the clay layers occurred. This phenomenon was confirmed by the analysis of the X-ray diffraction patterns of the composite, where the original basal peak of the clays decreased or completely disappeared, and the optimal distribution of the filler was observed using the transmission mode of light microscopy. Functional property testing showed that the composites have good antibacterial activity against Staphylococcus aureus, and the biocompatibility prediction demonstrated the formation of Ca- and P-containing particles through an in vitro experiment, thus applicable for medical use.


Introduction
Research on hybrid inorganic-organic nanocomposites has intensified in recent decades. Nanocomposites of polymers with pure or modified clay minerals are of particular interest. Minerals from the smectite group are more often used as nanofillers in polymer composites than minerals from the vermiculite group. In general, the addition of nanoclays to a polymer can be a way to improve many of the polymer's shortcomings, such as the mechanical properties of stiffness, tensile strength, and toughness or the barrier properties of permeability to gases and moisture, which is useful in applications such as food packaging. A similar positive effect is observed in improved fire resistance or an increase in the thermal stability of the polymer, which allows it to withstand higher temperatures. These properties may vary depending on the type and amount of clay used, as well as the processing conditions of the nanocomposite [1].
Polymer composites are widely used in medicine, for example, for bone tissue engineering [2], as surgical devices (miniscrews) [3], biomaterials [4], implant coatings [5], etc. Implanted devices are often associated with bacterial infection, which poses a significant threat to patients. High infection rates (2-6%) are observed in orthopedic implants, dental techniques, can then be employed to process these biomaterials into suitable forms for bone-tissue engineering [26].
Our study was focused on the laboratory preparation of a polymer nanocomposite with an antibacterial and biocompatible clay hybrid nanofiller. The aim of the work was to obtain the desired functional properties by combining polyethylene with vermiculite or montmorillonite filler, which was intercalated with chlorhexidine diacetate and then modified with Ca-deficient hydroxyapatite. The structures of the final polymer nanocomposites were studied using X-ray diffraction (XRD) analysis, infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). The particle size distribution of the clay nanofillers was measured and observed using light microscopy and laser scattering methods. Finally, the antibacterial activity against Gram-positive Staphylococcus aureus was studied. Biocompatibility tests, according to Kokubo [27], were performed by soaking polymer nanocomposites in simulated body fluids (SBF).

Materials and Synthesis
The synthesis of the composite consisted of three steps: intercalation-decorationcompounding.
The composite of clay mineral/CA/CDH was prepared according to our previous study [19]. Firstly, vermiculite (V) or montmorillonite (M) were intercalated with chlorhexidine diacetate (CA) using a cation exchange process in a wet process (VCA or MCA, respectively). The next step was the decoration of the intercalated clays with CDH particles. The CaCl 2 solution was slowly added to a solution of Na 2 HPO 4 , which contained VCA or MCA. The precipitate was allowed to sediment for 24 h. After this period, the supernatant was decanted, and the precipitate was dried at 70 • C for 12 h. The samples were marked as MCAH (for MCA + CDH) and VCAH (for VCA + CDH).
Polyethylene nanocomposites were prepared from the mixtures containing 38.8 g of LDPE mixture and 1.2 g (3 wt. %) of nanofillers MCAH and VCAH, respectively. Each mixture was blended in the Brabender single screw extruder 19/25 kneading chamber (Brabender GmbH & Co., Duisburg, Germany) at 160 • C for 10 min in two rate intervals (10 rpm for 2 min and subsequently 50 rpm for 8 min). Then, the matter was pressed at 160 • C into the 1 mm thick plates of size 100 mm × 100 mm. The polyethylene (PE) composites were marked as VCAH/PE and MCAH/PE.

Methods for Characterization
The composites were studied employing X-ray powder diffraction (XRD) methods using an X-ray diffractometer Rigaku Ultima IV (Tokyo, Japan) (reflection mode, Bragg-Brentano arrangement, CuKα 1 radiation, working condition 40 kV, 40 mA) in the ambient atmosphere under constant conditions. The phase analysis of the studied samples was performed based on the Database JCPDS ICDD PDF4+(2022).
The images of samples were performed by scanning electron microscopy (SEM) on a PHILIPS XL-30 (FEI, Hillsboro, OR, USA) equipped with an energy dispersive spectrometer (EDS). The samples were coated with gold/palladium to ensure the good conductivity for surface examination, and a secondary electrons detector was used.
The differential scanning calorimetry (DSC) using Setaram DSC 131 evo (Caluire, France) calorimeter (from −25 to 150 • C, 5 • /min, Ar atmosphere) was used to study the thermal properties of the composites. The images of the composite were acquired using light microscopy (LM) on an Olympus BX51 (Tokyo, Japan) equipped with a camera Olympus UC30, using bright field polarized light at transmission mode.
The particle size of the clay hybrid filler was measured using sizer HORIBA Partica LA-950 (Tokyo, Japan) in distilled water (used refractive indexes for water 1.33, vermiculite 1.54, montmorillonite 1.49).

Antibacterial Tests
The antibacterial activity was studied against Gram-positive Staphylococcus aureus. After 24 h, 48, 72, and 96 h incubation under similar conditions, the active bacteria were counted.
An antibacterial test of the prepared LDPE nanocomposites was performed by the microbial fingerprints technique. The method assumes that the bacteria under the same conditions gradually die. Each sample plate was cut to the three squared plates (25 cm 2 ). Then, 100 µL of the bacterial suspension of Gram-positive S. aureus CCM 3953 (1.0 × 10 6 cfu.mL −1 ), provided by the Czech collection of microorganisms (CCM), was spread on the plates and was left to dry in the laminar box at 21 • C. Then, the dried bacterial suspension on the surface of plates was stamped using the microbial fingerprints technique on the three discs with blood agar in 24, 48, 72 and 96 h time intervals. The bacteria cultivation took place in the thermostat at 35 • C for 24 h. The number of colony-forming units of bacteria (CFU) at all three fingerprints were counted and averaged.

In Vitro Biocompatibility
The in vitro biocompatibility test was carried out according to Kokubo et al. [27] by soaking the samples in simulated body fluid (SBF) solution.
The required amount of SBF solution for each sample is calculated as follows: where V s is the volume of the SBF (mL), and S a is the apparent surface area of the specimen (mm 2 ). The calculated volume of the SBF was put into a plastic bottle. After heating the SBF to 36.5 • C, a specimen was placed in the SBF so that the sample was hanging in the bulk of the testing container, not touching the walls of the container. The solution was changed every week for simulation of the dynamic conditions in the human body, and after soaking for a period of 4 weeks in the SBF, the specimens were removed from the SBF and gently washed with pure water. Then, the samples were dried at laboratory temperature and placed in a desiccator [27].
The formation of the layer on the surface of the immersed samples was investigated using a scanning electron microscope.

Results and Discussion
For all stages of sample preparation, the characterization of the properties and parameters was carried out. Figure 1A shows the monoionic Na-montmorillonite XRD pattern and Figure 1B(a) the patterns of pure polyethylene and as well as the fillers MCA and MCAH followed by the complete PE composites. The pure PE pattern presented mainly two intensive reflections at d (001) = 0.41 nm and d (200) = 0.38 nm, which corresponded to the orthorhombic structure of PE [28,29]. The PE pattern contained another weak reflection d = 0.46 nm evaluated as PDF card no. 00-011-0834. The filler MCA showed a diffused basal reflection of montmorillonite intercalated with chlorhexidine diacetate at d = 1.55 nm [12] corresponding to the lateral alignment of large CA molecules ( Figure 1B(b)). After decoration with CDH, the MCAH pattern showed in both phases a basal reflection of intercalated montmorillonite at  Figure 1B(d). The diffraction patterns of the PE composites showed minimal peak traces of intercalated MCA and regular peaks of PE in PE/MCA and PE/MCAH, as shown in Figure 1B(c,e), respectively. Based on this observation, we can estimate that montmorillonite exfoliation took place in both composites. uated as PDF card no. 00-011-0834. The filler MCA showed a diffused basal reflection of montmorillonite intercalated with chlorhexidine diacetate at d = 1.55 nm [12] corresponding to the lateral alignment of large CA molecules ( Figure 1B(b)). After decoration with CDH, the MCAH pattern showed in both phases a basal reflection of intercalated montmorillonite at d = 1.55 nm and basal reflections of CDH at d = 0.334 and 0.28 nm, as shown in Figure 1B(d). The diffraction patterns of the PE composites showed minimal peak traces of intercalated MCA and regular peaks of PE in PE/MCA and PE/MCAH, as shown in Figure 1B(c,e), respectively. Based on this observation, we can estimate that montmorillonite exfoliation took place in both composites.

X-ray Diffraction Phase Analysis
The polyethylene composite with MCAH filler exhibited intensive basal reflections of PE and a reflection of CDH at d = 0.28 nm ( Figure 1B(e)). The XRD patterns of the pure Na-vermiculite ( Figure 1A) and the set of PE composites and vermiculite fillers are shown in Figure 2B. The vermiculite intercalated with chlorhexidine diacetate showed two well-defined and intensive reflections at d = 2.933 nm and 2.14 nm [12] and residuum of the original non-intercalated peak. The PE/VCA showed a reflection of pure PE (d = 0.46 nm, 0.415 nm, and 0.376 nm) and intercalated vermiculite (d = 2.933 and 2.14 nm), where the intensity compared to the pattern in Figure 2B(b) was visibly lower. In this composite, the reflection of the PE remained without changes, and the intercalated vermiculite peaks positions were not changed; only the relative intensity of the V-peaks was lower ( Figure 2B(c)).
The filler VCAH presented a reflection of the intercalated vermiculite (d = 2.933 nm and 2.14 nm) in accordance with [30,31] and reflections of the CDH (d = 0.343 nm, 0.282 nm, and 0.199 nm) ( Figure 2B(d)). The intercalated vermiculite pattern peaks intensities were lower after modification with CDH.
Finally, the polymer composite PE/VCAH showed diffractions of pure PE and the reflection of CDH at 0.282 nm. The intercalated vermiculite was represented by a very low intensity peak at 2.14 nm ( Figure 2B(e)). This pattern is expressing the acknowledged exfoliation of the vermiculite silicate layers; however, the decoration of the vermiculite with CDH remained evident. The XRD patterns of the pure Na-vermiculite ( Figure 1A) and the set of PE composites and vermiculite fillers are shown in Figure 2B. The vermiculite intercalated with chlorhexidine diacetate showed two well-defined and intensive reflections at d = 2.933 nm and 2.14 nm [12] and residuum of the original non-intercalated peak. The PE/VCA showed a reflection of pure PE (d = 0.46 nm, 0.415 nm, and 0.376 nm) and intercalated vermiculite (d = 2.933 and 2.14 nm), where the intensity compared to the pattern in Figure 2B(b) was visibly lower. In this composite, the reflection of the PE remained without changes, and the intercalated vermiculite peaks positions were not changed; only the relative intensity of the V-peaks was lower ( Figure 2B(c)).  The filler VCAH presented a reflection of the intercalated vermiculite (d = 2.933 nm and 2.14 nm) in accordance with [30,31] and reflections of the CDH (d = 0.343 nm, 0.282 nm, and 0.199 nm) ( Figure 2B(d)). The intercalated vermiculite pattern peaks intensities were lower after modification with CDH.
Finally, the polymer composite PE/VCAH showed diffractions of pure PE and the reflection of CDH at 0.282 nm. The intercalated vermiculite was represented by a very low intensity peak at 2.14 nm (Figure 2B(e)). This pattern is expressing the acknowledged exfoliation of the vermiculite silicate layers; however, the decoration of the vermiculite with CDH remained evident.
The XRD pattern of the polymer composites confirmed the stable structure of the PE, the intercalation of the clay minerals with chlorhexidine diacetate, and the presence of CDH. The peak positions of all the components remained at the same values, indicating a lack of PE intercalation into the clay mineral interlayer or interaction with CDH.

Differential Scanning Calorimetry Structure Study
The crystallinity (Xc) of the polymer composites can be used to characterize the ratio of the crystalline part of the semi-crystalline polymer, and for the calculation of Xc, we used the following formula: where ∆Hm is the enthalpy of melting during the heating process, ∆H100 is tabulated as the enthalpy of the crystallization-melting process. The ∆H100 of polyethylene is 293 J/g [32]. The crystallinity of the composites was lower than that of the pure PE. Fillers affect the crystallinity of the PE matrix shifting the values to lower numbers. A polymer composite with montmorillonite filler affects the polyethylene composite crystallinity less than vermiculite fillers. The crystallization temperature of the polyethylene composites compared to the pure PE decreased, but the melting temperature remained the same. (see Table 1).

Light Microscopy Imagining and Particle Size Distribution
The distribution of the filler in PE matrix was studied using light microscopy imagining. Figure 3 shows the distribution of MCA in the PE matrix. The MCA particles in range 5-60 µm were dispersed in the PE matrix uniformly. The highest number of particles (44%) was in the range of 10-20 µm, the second most populated particles (32%) were in the range of 20-30 µm, the third were particles in the range of 30-40 µm at 17% (Figure 3).
In the case of the MCAH, the situation was different. The number of smaller particles was visibly higher-the amount was about 50% in the 10-20 µm range. The second in the range of 20-30 µm was higher than for the MCA at 21%. The third most common particles were in the 5-10 µm range. There were also particles in the range of 60-70 µm, which may be caused by the formation of large aggregates during the preparation of the polymer composite.

3).
In the case of the MCAH, the situation was different. The number of smaller particles was visibly higher-the amount was about 50% in the 10-20 μm range. The second in the range of 20-30 μm was higher than for the MCA at 21%. The third most common particles were in the 5-10 μm range. There were also particles in the range of 60-70 μm, which may be caused by the formation of large aggregates during the preparation of the polymer composite. The PE/VCA showed a larger particle distribution than the PE with montmorillonite fillers (Figure 4). The particles were mostly in the 30-40 μm range (35%), the second interval was 20-30 μm (31%), and the third interval included particles in the 10-20 μm range (27%). The presence of larger particles may appear during the preparation of the composite.
The PE/VCAH composite showed the lowest number of particles compared to other polymer composites but contained larger particles that were likely to form aggregates. This composite showed clusters of particles around 100 μm (1% of particles) (Figure 4).  The PE/VCA showed a larger particle distribution than the PE with montmorillonite fillers (Figure 4). The particles were mostly in the 30-40 µm range (35%), the second interval was 20-30 µm (31%), and the third interval included particles in the 10-20 µm range (27%). The presence of larger particles may appear during the preparation of the composite. range of 20-30 μm was higher than for the MCA at 21%. The third most common particles were in the 5-10 μm range. There were also particles in the range of 60-70 μm, which may be caused by the formation of large aggregates during the preparation of the polymer composite. The PE/VCA showed a larger particle distribution than the PE with montmorillonite fillers (Figure 4). The particles were mostly in the 30-40 μm range (35%), the second interval was 20-30 μm (31%), and the third interval included particles in the 10-20 μm range (27%). The presence of larger particles may appear during the preparation of the composite.
The PE/VCAH composite showed the lowest number of particles compared to other polymer composites but contained larger particles that were likely to form aggregates. This composite showed clusters of particles around 100 μm (1% of particles) (Figure 4).  The PE/VCAH composite showed the lowest number of particles compared to other polymer composites but contained larger particles that were likely to form aggregates. This composite showed clusters of particles around 100 µm (1% of particles) (Figure 4).
The distribution of fillers in all the polyethylene matrices was relatively homogeneous, which ensured the uniform presence of antibacterial and biocompatible parts on the entire surface of the studied polymer composites. In all the studied polymer composites, the particles were primarily in the range of 10-40 µm ( Figure 5).
The particle size distribution (PSD) results of the fillers before compounding to PE are presented in Table 2. All the fillers median value was in a relatively narrow range from 11 to 15 µm. Generally, both vermiculite fillers were more uniform in size, and the median was higher. The montmorillonite fillers showed a trimodal or bimodal distribution. The wide distribution of MCAH and VCAH is due to the presence of the CDH that forms aggregates with clay minerals, as discussed in our previous study [33]. These findings are in correlation with the LM observation of the filler size in the matrix; however, the effect of the plate-like particles should be considered. Looking at the images acquired via transmission mode, we see the "flat particles" but not the one arranged perpendicularly, while the PSD method considers the particle as a 3D object. Therefore, the average particle evaluated by the LM was larger than the one from the PSD.
The distribution of fillers in all the polyethylene matrices was relatively homogeneous, which ensured the uniform presence of antibacterial and biocompatible parts on the entire surface of the studied polymer composites. In all the studied polymer composites, the particles were primarily in the range of 10-40 μm ( Figure 5). Figure 5. Histogram of the filler particles counted using light microscopy observation.
The particle size distribution (PSD) results of the fillers before compounding to PE are presented in Table 2. All the fillers median value was in a relatively narrow range from 11 to 15 μm. Generally, both vermiculite fillers were more uniform in size, and the median was higher. The montmorillonite fillers showed a trimodal or bimodal distribution. The wide distribution of MCAH and VCAH is due to the presence of the CDH that forms aggregates with clay minerals, as discussed in our previous study [33]. These findings are in correlation with the LM observation of the filler size in the matrix; however, the effect of the plate-like particles should be considered. Looking at the images acquired via transmission mode, we see the "flat particles" but not the one arranged perpendicularly, while the PSD method considers the particle as a 3D object. Therefore, the average particle evaluated by the LM was larger than the one from the PSD.

Antibacterial Test
The antibacterial activity of the polymers containing clay mineral, hydroxyapatite, and chlorhexidine diacetate were compared with those containing only clay mineral and chlorhexidine. The surface of each sample was covered with a suspension of bacteria Staphylococcus aureus. The bacteria survived on the surface of clean PE plates throughout the experiment. The antibacterial activity of the MCAH was very low at the beginning of the experiment but increased rapidly after 96 h. In the case of the VCAH, the antibacterial effect was almost 50% and finally almost 60% ( Figure 6, Table 3). This composite had very similar antibacterial activity throughout the test. The results in Figure 6 show that the composites without hydroxyapatite showed better antibacterial behavior, which may be due to the coating of the clay mineral surface with hydroxyapatite. The increase in the CA content in clay minerals should be investigated in the future.

Antibacterial Test
The antibacterial activity of the polymers containing clay mineral, hydroxyapatite, and chlorhexidine diacetate were compared with those containing only clay mineral and chlorhexidine. The surface of each sample was covered with a suspension of bacteria Staphylococcus aureus. The bacteria survived on the surface of clean PE plates throughout the experiment. The antibacterial activity of the MCAH was very low at the beginning of the experiment but increased rapidly after 96 h. In the case of the VCAH, the antibacterial effect was almost 50% and finally almost 60% ( Figure 6, Table 3). This composite had very similar antibacterial activity throughout the test. The results in Figure 6 show that the composites without hydroxyapatite showed better antibacterial behavior, which may be due to the coating of the clay mineral surface with hydroxyapatite. The increase in the CA content in clay minerals should be investigated in the future.

In Vitro Biocompatibility
The surface of the PE/MCAH before immersion in the SBF was relatively smooth; in some cases, the filler particles probably protruded from the polyethylene (Figure 7). After incubation in SBF, the surface of PE/MCAH was covered with irregular different shapes with a bumpy surface. Elements corresponding to the PE matrix (C), the apatite phase (Ca, P, O), and other elements originating from the SBF liquid, such as Na, Cl, Mg, were detected by EDS elemental analysis. The surface irregularities and filler particles that appeared on the polymer surface helped to create an apathetic structure from SBF because the CDH contained in the fillers allowed ions in the SBF to bond with the Ca or P ions from the CDH [34].  The surface of the PE/VCAH before and after immersion in SBF is shown in Figure 8. The surface of the PE/VCAH before immersion in SBF was relatively smooth with visible particles of fillers, which were not fully covered by the PE matrix. After immersion in the SBF, the surface was covered by a layer of small particles of apatite ( Figure 8b) and with large crystals of NaCl or MgCl2 (Figure 8c). The EDS analysis confirmed elements from SBF, such as Na, Mg, Cl, and Ca. The surface of the PE/VCAH before and after immersion in SBF is shown in Figure 8. The surface of the PE/VCAH before immersion in SBF was relatively smooth with visible particles of fillers, which were not fully covered by the PE matrix. After immersion in the SBF, the surface was covered by a layer of small particles of apatite ( Figure 8b) and with large crystals of NaCl or MgCl 2 (Figure 8c). The EDS analysis confirmed elements from SBF, such as Na, Mg, Cl, and Ca.

Conclusions
In the study, the structure, the morphology, and antibacterial/biocompatible properties of the newly prepared polymer composite with hybrid organic-inorganic clay nanofiller were investigated. Polymer composites were investigated for their structural properties when clay nanofillers were intercalated with chlorhexidine, where intercalation was confirmed with an XRD peak shift. The next step of the CDH decoration of the intercalated silicate layers was confirmed through the independent peaks of the CDH phases d = 0.34, 0.28, and 0.2 nm. After mixing with PE, the clay nanofiller delaminated, and the reflection of the clays was not visible in the XRD pattern. Observing the clay particles' distribution and homogenization in the PE matrix, we can state that montmorillonite dispersed better, creating smaller particles about 13 µm with more intensive exfoliation. The crystallinity of the composites during the temperature testing varied based on the filler type; vermiculite caused a lower crystallinity of the composite Xc = 33% compared to the one with montmorillonite filler Xc = 37%. Pure polyethylene was observed to show very poor antibacterial resistance, as the Staphylococcus aureus strain survived throughout the experiment on the tested surface. Nanoclay with chlorhexidine diacetate improved the antimicrobial properties and the subsequent modification of the clay with an inorganic biogenic compound (CDH) improved the biocompatible properties of the PE. The composite containing MCAH showed better antibacterial properties than the composite with VCAH; the biocompatibility properties looked very similar for both composites; so, the PE/MCAH appears to be the best candidate for further detailed study. Montmorillonite has the ability to intake a higher amount of intercalants to the structure; therefore, the antimicrobial ef-

Conclusions
In the study, the structure, the morphology, and antibacterial/biocompatible properties of the newly prepared polymer composite with hybrid organic-inorganic clay nanofiller were investigated. Polymer composites were investigated for their structural properties when clay nanofillers were intercalated with chlorhexidine, where intercalation was confirmed with an XRD peak shift. The next step of the CDH decoration of the intercalated silicate layers was confirmed through the independent peaks of the CDH phases d = 0.34, 0.28, and 0.2 nm. After mixing with PE, the clay nanofiller delaminated, and the reflection of the clays was not visible in the XRD pattern. Observing the clay particles' distribution and homogenization in the PE matrix, we can state that montmorillonite dispersed better, creating smaller particles about 13 µm with more intensive exfoliation. The crystallinity of the composites during the temperature testing varied based on the filler type; vermiculite caused a lower crystallinity of the composite Xc = 33% compared to the one with montmorillonite filler Xc = 37%. Pure polyethylene was observed to show very poor antibacterial resistance, as the Staphylococcus aureus strain survived throughout the experiment on the tested surface. Nanoclay with chlorhexidine diacetate improved the antimicrobial properties and the subsequent modification of the clay with an inorganic biogenic compound (CDH) improved the biocompatible properties of the PE. The composite containing MCAH showed better antibacterial properties than the composite with VCAH; the biocompatibility properties looked very similar for both composites; so, the PE/MCAH appears to be the best candidate for further detailed study. Montmorillonite has the ability to intake a higher amount of intercalants to the structure; therefore, the antimicrobial effect can be increased by the amount of CA, and the dispersibility of the filler can be improved.